This program advances an innovative approach that directly quantifies the impact of material properties on protein folding stability in situ at sub-micron spatial resolution and at millisecond time resolution. A major challenge in engineering hybrid biomaterials with protein components is that many materials contribute to substantial losses of protein activity, with significant commercial, scientific, and clinical ramifications. A major roadblock to solving this problem is the inability to quantify in situ the material properties that preserve or shut down protein function. This program will develop a dynamic fluorescence imaging approach, Fast Relaxation Imaging (FReI) that promises to overcome this roadblock and open the door to novel investigations of protein-material interactions, at the protein level. This program builds on our recent demonstration that FReI measurements are able to quantify protein folding stability in polyacrylamide hydrogels. We now seek to extend this approach to studies of biomedically relevant proteins and materials. Specific Aim 1 will test the capacity to identify physical chemical mechanisms by which materials perturb protein stability and function, by using two-dimensional substrates of controlled surface chemistry. Specific Aim 2 will extend studies to encapsulated proteins in three-dimensional alginate hydrogels, which are extensively used for drug delivery and tissue engineering. Outcomes from this program and anticipated subsequent research will lay the foundation for generating new molecular level design rules for robust, functional biomaterials with bio-macromolecular components.